Tuesday, April 24, 2012

The Krebs cycle
is a metabolic pathway composed of 8 biochemical reactions, each catalyzed by a
different enzyme. Here
is some information about the first four enzymes of the Krebs cycle ...

Citrate synthase

The
citrate synthase is an enzyme widely used as a biomarker for the presence of
intact mitochondria in cell cultures or organelle preparations. Despite
being a mitochondrial enzyme it is encoded by nuclear DNA and synthesized in
the cytosol.

This
enzyme is the first regulatory enzyme in the Krebs cycle. It uses
two different substrates, the acetyl-CoA and oxaloacetate. The
oxaloacetate firstly binds to the enzyme, which induces conformational changes
that create the binding site for the acetyl-CoA molecule.

From
a structural point of view, it is composed of 437 amino acid residues and has
two subunits, each with about 20 alpha helices. The
active center has three amino acid residues essential for the catalytic
function of the enzyme, due to the establishment of specific interactions with the
substrates - His274, His320, and Asp-375.

Its
mechanism of action involves an aldol condensation. To
view a video about the mechanism of action of citrate synthase, click here.

Aconitase

The
aconitase is an enzyme that has a functional iron-sulfur cluster [Fe4S4]2+,
which interacts with three cysteine ​​residues of the enzyme. It
is especially sensitive to oxidative stress and, in particular, to superoxide
anion, due to the iron-sulfur cluster.

It
has two homologues in our body, the iron-responsive element-binding protein
(IRE-BP) and the 2-isopropylmalate dehydratase (or alpha-isopropylmalate
isomerase).

From
a structural standpoint, the aconitase has two conformations, one for the
inactive and one for the active state. In
the inactive form, it has four domains, the first three establish interactions
with the iron-sulfur cluster, while the latter has the active center. When
it becomes active, the enzyme is altered in the iron-sulfur cluster (Fe3S4
turns in Fe4S4), and this represents the main difference
between the two conformations of the enzyme.

Its
mechanism of action relies on a mechanism of dehydration-hydration, via the
intermediate cis-aconitate.

Its active site has two amino acid residues particularly important for
catalytic activity - His101 and Ser642.

The
importance of this enzyme, in a physiological point of view, is supported by
the existence of many diseases that affect it. One is referred to
as aconitase deficiency. It
is caused by a mutation in the gene that codes for a protein responsible for
the assembling of the iron-sulfur cluster. This
disease causes myopathy and exercise intolerance, because the aerobic
catabolism of these individuals is compromised. Another
disease is Friedreich's ataxia (FRDA), characterized by a lower activity of
aconitase and other Krebs cycle enzyme, the succinate dehydrogenase. Besides
these, there are studies that suggest a possible relationship between aconitase
and diabetes. However,
it is still an hypothesis that has to be best characterized.

Isocitrate dehydrogenase

Isocitrate
dehydrogenase is the second regulatory enzyme in the Krebs cycle. There are
three different isoforms of isocitrate dehydrogenase. One
exists only in the mitochondrial matrix and uses NAD+ as the acceptor
of electrons. The
other isoforms use NADP+ as the acceptor of electrons and appear to
have as main function the formation of NADPH, essential for the reducing
anabolic reactions. These
forms are present in the mitochondrial matrix, the cytosol and in the
peroxisome.

The
forms using NADP+ as a cofactor have an homodimeric structure, while
the one that uses NAD+ is a heterotetramer.

The
reaction catalyzed by isocitrate dehydrogenase involves the formation of an
intermediary, the oxalossuccinate.

From
the clinical point of view, some mutations were found in isocitrate
dehydrogenase in some brain tumors, including astrocytoma, oligodendroglioma
and multiforme glioblastoma. There
are also some studies that indicate a possible relationship between mutations
in the enzyme and acute myeloid leukemia.

Alpha-ketoglutarate dehydrogenase

This
is the third (and last!) regulatory point of the Krebs cycle.

This
enzyme, which can also be referred to as oxoglutarate dehydrogenase, is
actually a multienzyme complex. It
consists of the following enzymes: alpha-ketoglutarate dehydrogenase, dihydrolipoyl
succinyltransferase dihydrolipoyl dehydrogenase. It
has a structure and a reaction mechanism very similar to the pyruvate
dehydrogenase complex. Because
of this, it is believed that possibly both complexes had a common origin and at
some point of evolution they suffered a divergent evolution.

Clinically,
this enzyme complex functions as an autoantigen in primary biliary cirrhosis, a
form of acute hepatic failure. Moreover,
its catalytic activity is also decreased in various neurodegenerative diseases.